Studies have shown that vitamin D can enhance glucose-stimulated insulin secretion (GSIS) and change the expression of genes in pancreatic β-cells. Still the mechanisms linking vitamin D and GSIS are unknown. We used an established β-cell line, INS1E. INS1E cells were pre-treated with 10nM 1,25(OH)2vitamin D or 10nM 25(OH)vitamin D for 72h and stimulated with 22mM glucose for 60min. RNA was extracted for gene expression analysis. Expression of genes affecting viability, apoptosis and GSIS changed after pre-treatment with both 1,25(OH)2vitamin D and 25(OH)vitamin D in INS1E cells. Stimulation with glucose after pre-treatment of INS1E cells with 1,25(OH)2vitamin D resulted in 181 differentially expressed genes, whereas 526 genes were differentially expressed after pre-treatment with 25(OH)vitamin D. Vitamin D metabolites may affect pancreatic β-cells and GSIS through changed gene expression for genes involved in β-cell function and viability.